Adipose tissue lipoprotein lipase (LPL) hydrolyses the core of triglyceride-rich lipoproteins into free fatty acids. LPL is important for the generation of HDL2, and is central to the pathogenesis of hypertriglyceridemia and atherosclerosis. In previous studies, only LPL bioactivity could be measured, and neither the LPL protein nor mRNA could be measured directly. The candidate has developed a system of isolated human adipocytes that produce LPL in culture and with the help of his collaborators, the candidate has developed techniques to quantitate LPL immunoreactive mass and LPL mRNA levels. The specific aims of this project are as follows: 1) What is the pathway of LPL biogenesis? 2) Is LPL initially synthesized as an inactive or partially active precursor? 3) Is LPL regulated in vitro by such potential regulators as glucose, insulin, IGF1, adenosine, prostaglandin E2, tumor necrosis factor and catecholamines? 4) What is the mechanism of such regulation? 5) Can LPL mRNA be identified and quantitated? 6) By what mechanism do metabolic disorders such as obesity, diabetes, and hypertriglyceridemia affect LPL? To approach question 1, LPL will be radiolabeled and immunoprecipitated in the presence of inhibitors of glycoprotein processing and the LPL precursors that are formed will be identified by SDS-PAGE and autoradiography. To determine whether these precursors are bioactive (question 2), LPL activity will be measured and specific activity will be determined by measuring LPL immunoreactive mass in an ELISA. To study regulation (question 3), LPL bio- and immunoactivity will be measured in cells exposed to each potential regulator listed above and the processing inhibitors will be used to block the effect of each regulatory substance (question 4). Using a cDNA probe to LPL developed by Drs. Kirschgessner and Schotz, LPL mRNA will be quantitated by Northern analysis and slot blotting (question 5). Finally adipose tissue LPL activity, immunoreactive mass, and mRNA levels will be quantitated in patients with obesity, diabetes, and hypertriglyceridemia before and after treatment, and in normal subjects before and after feeding (question 6). Assisting the candidate in this project are Dr. Michael Schotz, Dr. Todd Kirschgessner, and Dr. John Goers, who are supplying the cDNA probe, the anti-LPL antibodies, as well as advice and expertise.